The goal of our research group is to elucidate the molecular mechanisms underlying the initiation phase of protein synthesis in eukaryotic organisms. We use the yeast saccharomyces cerevisiae as a model system and employ a range of approaches - from genetics to biochemistry to structural biology - in collaboration with Alan Hinnebusch and Tom Devers labs at NICHD and several other research groups around the world. Eukaryotic translation initiation is a key control point in the regulation of gene expression. It begins when an initiator methionyl tRNA (Met-tRNAi) is loaded onto the small (40S) ribosomal subunit. Met-tRNAi binds to the 40S subunit as a ternary complex (TC) with the GTP-bound form of the initiation factor eIF2. Three other factors eIF1, eIF1A and eIF3 also bind to the 40S subunit and promote the loading of the TC. The resulting 43S pre-initiation complex (PIC) is then loaded onto the 5-end of an mRNA with the aid of eIF3 and the eIF4 group of factors the RNA helicase eIF4A; the 5-7-methylguanosine cap-binding protein eIF4E; the scaffolding protein eIF4G; and the 40S subunit- and RNA-binding protein eIF4B. Both eIF4A and eIF4E bind to eIF4G and form the eIF4F complex. Once loaded onto the mRNA, the 43S PIC is thought to scan along the mRNA in search of an AUG start codon. This process is ATP-dependent and likely requires multiple RNA helicases, including the DEAD-box protein Ded1p. Recognition of the start site begins with base pairing between the anticodon of tRNAi and the AUG codon. This base pairing then triggers downstream events that commit the PIC to continuing initiation from that point on the mRNA. These events include ejection of eIF1 from its binding site on the 40S subunit, movement of the C-terminal tail (CTT) of eIF1A, and release of phosphate from eIF2, which converts it to its GDP-bound state. In addition, the initiator tRNA moves from a position that is not fully engaged in the ribosomal P site (termed P(OUT)) to one that is (P(IN)) and the PIC as a whole converts from an open conformation that is conducive for scanning to a closed one that is not. At this stage eIF2GDP dissociates from the PIC and eIF1A and a second GTPase factor, eIF5B, coordinate joining of the large ribosomal subunit to form the 80S initiation complex. eIF5B hydrolyzes GTP, which appears to result in a conformational reorganization of the complex, and then dissociates along with eIF1A. This year we have advanced our understanding of how the translation initiation factor eIF4A promotes recruitment of mRNA to the eukaryotic PIC. Our data indicate that ATP hydrolysis by the factor accelerates the rate of mRNA recruitment regardless of the level of structure in the message. In addition, the PIC stimulates the rate of ATP hydrolysis by the factor, suggesting a functional interaction between the two. Structures in the 5-UTR and 3 of the start codon in model mRNAs synergistically inhibit mRNA recruitment and their effects are both relieved by eIF4A, indicating that the factor can modulate structure in mRNA beyond what is present in the 5-UTR itself. Taken together, our data suggest that eIF4A might act on the PIC to mediate conformational changes required for loading of mRNA into the entry channel of the 40S ribosomal subunit in addition to its role in breaking up structures in the message itself. We have also made progress in studies of the related DEAD-box RNA helicase Ded1. Our data indicate that Ded1 is important for unwinding stable structures in mRNA 5-UTRs in collaboration with the RNA-binding and scaffolding protein eIF4G, but that it acts in mechanistically distinct fashions on different mRNAs. Finally, our genome-wide analyses of modulation of start codon usage by external factors has indicated a surprising diversity in responses of individual mRNAs, suggesting that multiple mechanisms can control this process.